Journal: Journal of Inflammation Research

Article Title: Endothelin-1/Endothelin Receptor Type A-Angiopoietins/Tie-2 Pathway in Regulating the Cross Talk Between Glomerular Endothelial Cells and Podocytes in Trichloroethylene-Induced Renal Immune Injury

doi: 10.2147/jir.s301104

Figure Lengend Snippet: Figure 10 Protein levels of podocin and nephrin. (A) showed the Western blot results of renal podocin and nephrin. (B and C) showed the optical density values of podocin and nephrin, respectively. Compared to solvent control group, podocin and nephrin were significantly decreased in TCE sensitized positive mice (P<0.05). Podocin and nephrin increased in TCE+BQ123 sensitized positive mice compared to TCE sensitized positive mice (P<0.05). *P<0.05.

Article Snippet: Anti-goat polyclonal Tie2 antibody, anti-goat polyclonal nephrin antibody, antigoat polyclonal Ang-1 antibody, anti-rabbit polyclonal phosphor-Tie-2 (pTie-2) antibody and human ET-1 ELISA kit were acquired from R&D Systems (Minnesota, USA).

Techniques: Western Blot, Solvent, Control

Journal: bioRxiv

Article Title: APOL1 is not expressed in proximal tubules and is not filtered

doi: 10.1101/623918

Figure Lengend Snippet: A . Immunofluorescent staining for APOL1, Nephrin, and CD31 in the BAC-APOL1 transgenic mouse kidney (G1 mouse is shown). Scale bar=40µm. B-C . Serial sections of BAC-APOL1 mouse kidney for ( B ) APOL1 protein using an immunohistochemical stain (brown, no counterstain) and for ( C ) APOL1 mRNA expression using in situ hybridization (blue, no counterstain) confirming expression in kidney was restricted to podocyte and endothelium. D . Proteinuria in single and dual transgenic mice (coomassie stain, arrowhead marks albumin). Additional low molecular weight urinary proteins (asterisk) are a normal finding in mice. E . Western blot of mouse serum in the same single and dual transgenic mice showing maintenance of high levels of serum APOL1 protein in the setting of proteinuria. G0 sera were diluted 1:10, G1 and G2 sera were undiluted. APOA1 Western blot as a positive control for serum loading, all were diluted 1:10. Normal concentrations of APOL1 (42.5kDa) in human sera range from 3-30µg/ml. Normal APOA1 (28kDa) concentrations in human and mouse sera are similar and range from 0.9-2.0µg/ml.

Article Snippet: Primary antibodies used include rabbit anti-human APOL1 (Sigma, HPA018885, lot E105260, 1:400 dilution), rabbit anti-mouse APOA1 (ThermoFisher, 1:500 dilution), goat anti-mouse CD31 (R&D Systems, 1:200 dilution), guinea pig anti-Nephrin (USB, 1:200).

Techniques: Staining, Transgenic Assay, Immunohistochemical staining, Expressing, In Situ Hybridization, Molecular Weight, Western Blot, Positive Control

Journal: Journal of Clinical Investigation

Article Title: Shifts in podocyte histone H3K27me3 regulate mouse and human glomerular disease

doi: 10.1172/jci95946

Figure Lengend Snippet: Figure 3. Inhibition of Jmjd3 and UTX increases podocyte H3K27me3 levels and attenuates Notch-mediated dedifferentiation. (A) Immunoblotting for H3K27me3 in cultured mouse podocytes treated with the Jmjd3 and UTX inhibitor GSK-J4 (5 μM for 48 hours) (n = 3). (B and C) Effect of GSK-J4 on podocyte Jagged-1 mRNA (n = 3) (B) and protein (n = 5) (C) levels. (D) N1-ICD levels in mouse podocytes exposed to recombinant TGF-β1 (10 ng/ml for 48 hours) in the presence or absence of GSK-J4 (5 μM) (n = 3). (E and F) α-SMA (E) (n = 6) and podocin (F) (n = 6) mRNA levels in podocytes exposed to TGF-β1, with or without GSK-J4. (G) Immunoblotting for H3K27me3 in kidney homogenates of mice treated with GSK-J4 (10 mg/kg thrice weekly i.p. for 10 days) (n = 3). (H) Immu- nofluorescence to detect the podocyte marker nephrin and H3K27me3 in glomeruli from mice treated with vehicle or GSK-J4 for 10 days, showing increased nuclear H3K27me3 levels in nephrin-positive glomerular cells (n = 3). DAPI staining is shown in blue. Original magnification, ×630. (I) Urine albumin/creati- nine ratio in BALB/c mice injected with adriamycin or vehicle and treated with GSK-J4 10 mg/kg thrice weekly i.p. or vehicle for 10 days (control plus vehicle, n = 6; control plus GSK-J4, n = 6; adriamycin plus vehicle, n = 6; adriamycin plus GSK-J4, n = 6). Values represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-tailed Student’s t test (A–C and G) and 1-way ANOVA followed by Fisher’s LSD post-hoc test (D–F and I).

Article Snippet: Glomerular TUNEL-positive nuclei were counted in an average of approximately 100 glomeruli per kidney section in 3 to 7 mice per group for adriamycin- and vehicletreated mice and 9 to 12 mice per group for db/m and db/db mice. clonal antibody, 1:100 (5246S; Cell Signaling Technology); N1-ICD rabbit polyclonal antibody, 1:100 (ab8925; Abcam); Jagged-1 rabbit polyclonal antibody, 1:100 (ab7771; Abcam); Alexa Fluor 488 donkey anti-rabbit secondary antibody, 1:100 (A21206, Thermo Fisher Scientific); nephrin goat polyclonal antibody, 1:100 (AF3159; R&D Systems); Alexa Fluor 555 donkey anti-goat secondary antibody, 1:200 (ab150134, Abcam); and cleaved caspase-3 rabbit polyclonal antibody, 1:100 (9661S; Cell Signaling Technology).

Techniques: Inhibition, Western Blot, Cell Culture, Recombinant, Marker, Staining, Injection, Control

Journal: Journal of Clinical Investigation

Article Title: Shifts in podocyte histone H3K27me3 regulate mouse and human glomerular disease

doi: 10.1172/jci95946

Figure Lengend Snippet: Figure 4. Inhibition of Jmjd3 and UTX attenuates renal injury in diabetic mice. (A) Body weight, (B) kidney weight, (C) blood glucose, (D) HbA1c, (E) urine volume, and (F) 24-hour urine albumin excretion in nondiabetic db/m mice and diabetic db/db mice treated with vehicle or GSK-J4 (10 mg/kg) i.p. thrice weekly for 10 weeks (db/m plus vehicle, n = 12; db/m plus GSK-J4, n = 11; db/db plus vehicle, n = 10; db/db plus GSK-J4, n = 9). (G) Transmission electron micrographs and podocyte foot process widths for db/m and db/db mice treated with vehicle or GSK-J4 (db/m plus vehicle, n = 4; db/m plus GSK-J4, n = 4; db/db plus vehicle, n = 4; db/db plus GSK-J4, n = 4). Arrows mark areas of foot process widening in the glomerulus of the vehicle-treated db/db mouse. Scale bars: 2 μm. (H) Immunofluorescence to detect the podocyte marker nephrin and Jagged-1 in glomeruli from db/m and db/db mice treated with vehicle or GSK-J4 (db/m plus vehicle, n = 12; db/m plus GSK-J4, n = 11; db/db plus vehicle, n = 9; db/db plus GSK-J4, n = 9). DAPI staining is shown in blue. Original magnification, ×630. Values represent the mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001, by 1-way ANOVA followed by Fisher’s LSD post-hoc test.

Article Snippet: Glomerular TUNEL-positive nuclei were counted in an average of approximately 100 glomeruli per kidney section in 3 to 7 mice per group for adriamycin- and vehicletreated mice and 9 to 12 mice per group for db/m and db/db mice. clonal antibody, 1:100 (5246S; Cell Signaling Technology); N1-ICD rabbit polyclonal antibody, 1:100 (ab8925; Abcam); Jagged-1 rabbit polyclonal antibody, 1:100 (ab7771; Abcam); Alexa Fluor 488 donkey anti-rabbit secondary antibody, 1:100 (A21206, Thermo Fisher Scientific); nephrin goat polyclonal antibody, 1:100 (AF3159; R&D Systems); Alexa Fluor 555 donkey anti-goat secondary antibody, 1:200 (ab150134, Abcam); and cleaved caspase-3 rabbit polyclonal antibody, 1:100 (9661S; Cell Signaling Technology).

Techniques: Inhibition, Transmission Assay, Immunofluorescence, Marker, Staining

Journal: Journal of Clinical Investigation

Article Title: Shifts in podocyte histone H3K27me3 regulate mouse and human glomerular disease

doi: 10.1172/jci95946

Figure Lengend Snippet: Figure 5. H3K27me3, Jagged-1, EZH2, Jmjd3, and UTX in normal human glomeruli and in glomerular disease. (A) Immunofluorescence to detect H3K27me3, the podocyte marker nephrin, and Jagged-1 in human kidney tissue (control nephrectomy, n = 12; diabetic nephropathy nephrectomy, n = 12; control biopsy, n = 9; FSGS biopsy, n = 10). In normal kidneys, H3K27me3 was present in the nuclei of numerous glomerular cells (and tubule epithelial cells), including nephrin-positive cells (podocytes, indicated by thick white arrows). In glomerular disease (diabetic nephropathy or FSGS), there was loss of H3K27me3, including from podocytes (thin white arrows), especially in more severely scarred glomeruli, that coincided with Jagged-1 expression and nephrin downregulation. The zoomed image is an enlargement of the outlined area. DAPI staining is shown in gray. (B) Immunofluorescence images for EZH2, Jmjd3 (control nephrectomy, n = 6; diabetic nephropathy nephrectomy, n = 6; control biopsy, n = 6; FSGS biopsy, n = 6) or UTX (control nephrectomy, n = 5, diabetic nephropathy nephrectomy, n = 5; control biopsy, n = 8; FSGS biopsy, n = 8) and nephrin in normal human glomeruli and in glomeruli from individuals with diabetic nephropathy or FSGS. Despite elevated podocyte H3K27me3 levels, EZH2 was absent from podocyte nuclei in control glomeruli (arrowheads) and glomeruli from patients with diabetic nephropathy or FSGS. In contrast, Jmjd3 was expressed by many glomerular cells, including podo- cytes (gray arrows), and its expression was unaltered in glomerular disease. UTX was also expressed in normal human podocytes, and its expression was significantly increased in podocytes from individuals with diabetic nephropathy or FSGS. The zoomed images are an enlargement of the outlined areas. DAPI staining is shown in blue. Original magnification, ×200. (C–E) Quantification of podocyte H3K27me3 (C), Jmjd3 (D), and UTX (E) levels. Because UTX is considered to escape X chromosome inactivation, the podocyte UTX levels in females are highlighted in red. Values represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.01, by 2-tailed Student’s t test. Bx, biopsy; Nx, nephrectomy.

Article Snippet: Glomerular TUNEL-positive nuclei were counted in an average of approximately 100 glomeruli per kidney section in 3 to 7 mice per group for adriamycin- and vehicletreated mice and 9 to 12 mice per group for db/m and db/db mice. clonal antibody, 1:100 (5246S; Cell Signaling Technology); N1-ICD rabbit polyclonal antibody, 1:100 (ab8925; Abcam); Jagged-1 rabbit polyclonal antibody, 1:100 (ab7771; Abcam); Alexa Fluor 488 donkey anti-rabbit secondary antibody, 1:100 (A21206, Thermo Fisher Scientific); nephrin goat polyclonal antibody, 1:100 (AF3159; R&D Systems); Alexa Fluor 555 donkey anti-goat secondary antibody, 1:200 (ab150134, Abcam); and cleaved caspase-3 rabbit polyclonal antibody, 1:100 (9661S; Cell Signaling Technology).

Techniques: Immunofluorescence, Marker, Control, Expressing, Staining

Journal: Journal of Clinical Investigation

Article Title: Shifts in podocyte histone H3K27me3 regulate mouse and human glomerular disease

doi: 10.1172/jci95946

Figure Lengend Snippet: Figure 7. In mice with established glomerular disease, inhibition of Jmjd3 and UTX prevents both further podocyte H3K27me3 loss and albuminuria progression. (A) Immunoblotting for EZH2 in lysates of immortalized mouse podocytes after treatment with adriamycin (300 ng/ml) for 48 hours (n = 6). (B) Immunofluorescence for the podocyte protein nephrin and EZH2 in BALB/c mice 4 days after injection with vehicle (n = 4) or adriamycin (n = 4, 10 mg/ kg) and quantification of podocyte EZH2 levels. (C) Immunofluorescence staining for H3K27me3 and the podocyte marker nephrin in kidney sections from a normal control BALB/c mouse and from BALB/c mice 4 days and 12 days after adriamycin injection and from a BALB/c mouse 12 days after adriamy- cin injection and treatment with GSK-J4 (10 mg/kg i.p.) beginning on day 4. DAPI staining is shown in blue. Original magnification, ×630. (D) Podocyte H3K27me3 content (control, n = 4; adriamycin day 4, n = 4; adriamycin day 12, n = 4; adriamycin plus GSK-J4, n = 4). (E) Urine albumin/creatinine ratio in BALB/c mice injected with adriamycin and treated with GSK-J4 (10 mg/kg) i.p. thrice weekly or with vehicle for 8 days, beginning 4 days after adriamycin injection (vehicle, n = 6; GSK-J4, n = 7). Arrows indicate GSK-J4 injections. Values represent the mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001, by 2-tailed Student’s t test (A, B, and E) and 1-way ANOVA followed by Fisher’s LSD post-hoc test (D).

Article Snippet: Glomerular TUNEL-positive nuclei were counted in an average of approximately 100 glomeruli per kidney section in 3 to 7 mice per group for adriamycin- and vehicletreated mice and 9 to 12 mice per group for db/m and db/db mice. clonal antibody, 1:100 (5246S; Cell Signaling Technology); N1-ICD rabbit polyclonal antibody, 1:100 (ab8925; Abcam); Jagged-1 rabbit polyclonal antibody, 1:100 (ab7771; Abcam); Alexa Fluor 488 donkey anti-rabbit secondary antibody, 1:100 (A21206, Thermo Fisher Scientific); nephrin goat polyclonal antibody, 1:100 (AF3159; R&D Systems); Alexa Fluor 555 donkey anti-goat secondary antibody, 1:200 (ab150134, Abcam); and cleaved caspase-3 rabbit polyclonal antibody, 1:100 (9661S; Cell Signaling Technology).

Techniques: Inhibition, Western Blot, Immunofluorescence, Injection, Staining, Marker, Control

Journal: Nature communications

Article Title: A slit-diaphragm-associated protein network for dynamic control of renal filtration.

doi: 10.1038/s41467-022-33748-1

Figure Lengend Snippet: Fig. 1 | Subcellular localization and interactome of Nephrin and Neph1 in renal podocytes. A Epitope-localization of the anti-Nephrin/Neph1 ABs used for meAP- MS analysis (further details in Supplementary Fig. 2); inset: Micrograph taken by scanning-EM of glomerular podocytes from rat. Scale bar is 5 µm. Result is repre- sentative of at least 100 experiments. B Electron micrographs illustrating the dis- tribution of Nephrin molecules labeled by anti-Nephrin AB a2 (and visualized by gold-particle coupled anti-IgG ABs, black dots) on the extracellular surface (E-face) of freeze-fracture replicas prepared from isolated glomeruli. Image on the right is the frame on the left at expanded scale; FP is foot process, SD is slit-diaphragm. Note that immuno-gold particles for Nephrin are only observed in the slit-area of the SD (arrows). Scale bars are 500 nm. Micrographs are representative of at least four experiments. C Two-dimensional gel separation of CL-47 solubilized mem- brane fractions prepared from isolated rat glomeruli, Western-probed with ABs

Article Snippet: After thorough washing, replicas were reacted either with 12 nm gold-coupled donkey anti-rabbit IgG (Podocin) or with 12 nm gold-coupled donkey anti-goat IgG (Nephrin) secondary antibodies (1:30, Jackson ImmunoResearch Europe, Cambridgeshire), diluted in blocking buffer, O/N at RT.

Techniques: Labeling, Isolation, Two-Dimensional Gel Electrophoresis, Western Blot